Self-association of Zn-insulin at neutral pH: investigation by concentration gradient--static and dynamic light scattering

Abstract

Equilibrium self-association of Zn-insulin at pH 7.0 was characterized over the range 0.3-5mg/mL by simultaneous measurement of static and dynamic light scattering. Analysis of static light scattering yielded a concentration-dependent weight-average molecular weight, and analysis of dynamic light scattering yielded a concentration-dependent intensity-average diffusion coefficient. The concentration dependence of both quantities may be accounted for to within experimental precision by a simple model, according to which the basic structural unit of Zn-insulin at concentrations exceeding 0.3mg/mL is a hexamer H. With increasing total protein concentration, hexameric protomers may self-associate in accordance with an isodesmic scheme in which a protomer may add to any prexisting oligomer H(n) to form H(n+1) with an invariant stepwise equilibrium association constant.

Figure 1

Scattering intensity in units of R/K_opt plotted as a function of the w/v concentration of Zn-insulin and scattering angle θ, prior to removal of statistical outliers and combination of data obtained at all angles for each protein concentration.

Figure 2

Normalized autocorrelation functions measured for the following insulin concentrations: 4.99 g/l (green), 2.52 g/l (red), and 0.48 g/l (blue), plotted together with curves calculated according to the best fit of equation (1) to the respective data set.

Figure 3

Chromatographic elution patterns obtained following injection of insulin solutions of 4 mg/ml (dashed) and 2 mg/ml concentration (solid), as monitored by intensity of 90° scattering.

Figure 4

Scattering intensity in units of R/K_opt plotted as a function of the concentration of Zn-insulin. Dotted curve is best fit of isodesmic equilibrium association model, calculated using equation (7) with best-fit values of M₁ and log K_a given in the text.

Figure 5

D_app plotted as a function of the concentration of Zn-insulin. Solid curve is the best fit of the isodesmic equilibrium association model, calculated using equations (5), (6), (8), and (9), with the best-fit values of M₁, log K_a, and D1 given in the text.

Figure 6

Semilog plot of the mass fraction of major association states, calculated according to the equilibrium scheme of Milthorpe et al [3] (blue curves) and the present work (black curves) plotted against insulin concentration. M* and H* represent the relative abundances of monomer and hexamer according to Milthorpe et al, and H, H₂, H₃, and H₄ represent the relative abundances of hexamer and the dimer, trimer, and tetramer of hexamers calculated according to the best-fit isodesmic association model. Solid lines are plotted over the range of experimental measurement. Dotted blue lines represent extrapolation to lower concentration, and the dotted red line represents a hypothetical “patch” between the highest concentration results of Milthorpe et al and the lowest concentration results obtained in the present work.

Figure 7